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Method for signal amplification and detection on target deoxyribonucleic acid (DNA) sequence at normal temperature

A technology of DNA sequence and signal amplification, which is applied in the field of single nucleotide polymorphism (SNP) typing and low-abundance point mutation detection, DNA sequence detection, and can solve problems such as complexity and insufficient discrimination

Inactive Publication Date: 2014-06-25
PEKING UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

However, the exonuclease III amplification system is less than 1.5 times distinguishing between matching and single-base mismatch DNA sequences.
[0009] In summary, most of the various nuclease-based DNA signal amplification systems reported so far are relatively complex, and require precise temperature control or coupling with other amplification techniques to achieve high sensitivity and immunity to single-base interference DNA sequences. ability to distinguish

Method used

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  • Method for signal amplification and detection on target deoxyribonucleic acid (DNA) sequence at normal temperature
  • Method for signal amplification and detection on target deoxyribonucleic acid (DNA) sequence at normal temperature
  • Method for signal amplification and detection on target deoxyribonucleic acid (DNA) sequence at normal temperature

Examples

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Effect test

Embodiment 1

[0053] Example 1

[0054] like figure 2 As shown in b), in this embodiment, the target object is a single strand of DNA. 5'-PO 4 The fluorescent group FAM in the single-stranded double-labeled fluorescent nucleic acid probe at the end is labeled on the base of the third nucleotide from the 5' end, and the quenching group BHQ1 is labeled at the 3' end. Prepare different concentrations of target DNA solutions respectively. The specific steps of detection are as follows:

[0055] In 1× ThermoPol buffer solution (20mM Tris-HCl+10mM (NH 4 ) 2 SO 4 +10mMKCl+2mM MgSO 4 , pH 8.825°C), mix the probe with different concentrations of target DNA, perform temperature-rising annealing (heat program 85°C 90s, 65°C 90s, 50°C 90s, 37°C 180s), add Lambda exonuclease, rapidly Measure the change of the fluorescence value of the solution with time.

[0056] In this embodiment, the designed probe sequence is as follows:

[0057] 5'-PO 4 -TCT(-FAM)CCACAGACACATACTCCA-BHQ1-3' (SEQ ID No. 1...

Embodiment 2

[0068] Example 2

[0069] In this example, the target is a single strand of DNA. 5'-PO 4 The fluorescent group FAM in the single-stranded double-labeled fluorescent nucleic acid probe at the end is labeled on the base of the third nucleotide from the 5' end, and the quenching group BHQ1 is labeled at the 3' end. The 2nd and 5th positions from the 5' end of the probe are mismatched with the target DNA single strand, and the rest of the bases are matched. There is also an interfering strand with a single base difference from the target DNA strand in the sample. The position of the single base corresponds to the 4th nucleotide from the 5' end of the probe, so the interfering strand and the probe are at the 2nd, 4th, and 5th nucleotides. Bits are mismatched.

[0070] This example contains two experiments:

[0071] (1) Differentiation degree of different types of mismatches

[0072] The discrimination of different types of mismatches was determined by changing the base type o...

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Abstract

The invention discloses a method for signal amplification and detection on a target deoxyribonucleic acid (DNA) sequence at normal temperature. A DNA signal amplification system is established by using Lambda excision enzyme and a double-tagging fluorescence nucleic acid probe, and the reaction velocity of the Lambda excision enzyme is improved by virtue of the synergistic effect of a fluorescence tagging radial and a mispairing basic group coexisting in a 5' ortho-position. By adopting the method, the target DNA sequence is rapidly, sensitively and specially detected at normal temperature, and convenience is brought for further large-scale and automatic detection. Besides, the method can be independently applied to signal amplification detection, and can be also used with the combination of other template amplification based DNA amplification techniques, such as rolling circle amplification (RCA) and selective polymerase chain reaction (PCR), and various requirements of DNA detection are met.

Description

technical field [0001] The invention relates to the field of DNA sequence detection, including the technical fields of quantitative detection of trace DNA, typing of single nucleotide polymorphism (SNP) and detection of low-abundance point mutations. Specifically, the present invention uses Lambda exonuclease and double-labeled fluorescent nucleic acid probes to establish a DNA signal amplification system to detect target DNA sequences rapidly, highly sensitively, and specifically at room temperature. Background technique [0002] DNA signal amplification refers to the multiple signal generation of a single target DNA sequence through biochemical reactions, thereby improving the detection sensitivity of the target DNA sequence. When the signal amplification system can selectively distinguish the target DNA sequence from the interfering DNA sequence with a single base difference and make the signal amplification factors of the two different, the amplification of the signal di...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/682C12Q2521/325C12Q2563/107
Inventor 赵美萍吴曈勃肖先金
Owner PEKING UNIV
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